Production and Characterization of ArAE Family Members including Putative Efflux Transporters (PETs) from Bacteria and Aluminium Activated Malate Transporters (ALMTs) from Plants

The focus of this study is proteins from the ArAE family, specifically two subfamilies: firstly, the ALMT family of plant membrane channels, which have numerous vital roles in plants, and secondly, the bacterial family first described as PET inner membrane exporters. Constructs were created for expression of firstly, the C-terminal domain (CTD) of wheat ALMT1 in E. coli and secondly, three full-length ALMTs (ALMT from wheat and ALMT5, ALMT9 from Arabidopsis) in Nicotiana benthamiana for structural and biochemical studies. Unfortunately, it appears that the CTD is not an independent soluble domain as originally thought, and this domain is now predicted to contain transmembrane helices, making it unsuitable for study in the manner planned. Similarly, production of full length ALMTs was unsuccessful as when extraction and purification was attempted, the protein degraded. Thirdly, a bacterial member of the ArAE family was expressed and characterised: AaeB from E. coli, along with its putative binding partner, AaeA. This was shown to form a complex with and be vital for the stability of AaeB. A strategy was devised for expression, solubilisation, and purification of these proteins and once they were obtained in pure form they were subjected to a range of biochemical and structural experiments. Microcrystals of AaeA were produced, towards a strategy for structural determination by X-ray crystallography. The first Electron Microscopy examination was performed on complexes of AaeB, and negative stain classes were produced. The first in silico homology model of AaeA has been produced and validated by CD spectroscopy, providing a range of insights. The complex formed by AaeA and AaeB has also been probed by crosslinking and SEC MALLS analysis, suggesting a 6:2 or 6:3 stoichiometry. Together this has furthered our understanding of this poorly characterised membrane protein family and provided a set of clones and protocols for future studies.